Project Summary Meibomian gland dysfunction (MGD) is one of the most prevalent ophthalmic conditions with millions of Americans suffering from the condition. In addition to burdensome symptoms, meibum and tear film alterations from MGD can impact ocular health by leading to ocular surface desiccation and an increased risk of inflammation or infectious disease and an abnormal ocular flora. Although these things are becoming better understood, the molecular basis for the mechanisms of lipid layer and tear film disruption are not well understood. Initial evidence from the literature and preliminary studies suggests that the O-acyl omega hydroxy fatty acids (OAHFAs) found in the meibum and tear film lipids are associated with increased in vivo lipid layer disruption and tear film thinning. The long-term goal of this research is to identify a molecular marker that predicts structural and functional changes in the meibum and tear film such that it may be considered a potential targeted therapeutic. Specific Aim #1 is targeted to quantitate and identify OAHFAs that are proposed to be decreased in the meibum and tear film in MGD. Quantitative differences will be determined using mass spectrometry, with class and species identification resultant from electrospray ionization (ESI) with a quadrupole time-of-flight (q-TOF) tandem MS for high resolution mass and structural information and SWATH analysis. Specific Aim #2 is targeted to determine the structural impact of the decrease in OAHFAs on the lipid layer of the tear film (e.g., reduced thickness, non-uniform lipid lens appearance); a high resolution microscope will be used to study the in vivo structure of the lipid layer. Specific Aim #3 will determine the functional impact of the disorganization of the lipid layer relative to increased evaporation of the aqueous tear film via rapid tear film thinning. In Specific Aim #3, a non-invasive interferometric optical system will be used to measure tear film thinning on the ocular surface in relation to Specific Aims #1 and #2.